離子束誘導(dǎo)天竺葵花色突變體的變異機(jī)理研究
發(fā)布時(shí)間：2019-05-22 22:54
【摘要】：花色作為觀賞花卉重要的經(jīng)濟(jì)指標(biāo),在植物性狀改良中成為首要培育目標(biāo)。本研究利用碳離子束輻照天竺葵幼嫩枝條,在輻照當(dāng)代篩選出一株穩(wěn)定遺傳的花色突變體LPM。以揭示離子束誘導(dǎo)天竺葵突變體花色變異的機(jī)理為目的,本研究通過對(duì)天竺葵野生型與突變體表型觀察、花色形成原因分析、類黃酮成分和含量鑒定以及花色形成關(guān)鍵結(jié)構(gòu)基因的差異表達(dá)分析,系統(tǒng)開展了離子束輻照后突變體花色變淺的機(jī)制研究。通過RNA-Seq高通量測(cè)序技術(shù)對(duì)花瓣轉(zhuǎn)錄組進(jìn)行深度測(cè)序,篩選野生型和突變體間差異表達(dá)的unigene,預(yù)測(cè)天竺葵花色形成相關(guān)基因,最終闡明離子束輻照引起天竺葵突變體花色改變的分子機(jī)理,為后續(xù)花色改良和新品種選育提供理論基礎(chǔ)。以下是本研究的主要內(nèi)容和結(jié)果:1、離子束30Gy輻照處理篩選到一株花色突變體LPM,與對(duì)照相比其花色和生理指標(biāo)均發(fā)生了明顯改變。解剖學(xué)觀察顯示RWT花瓣細(xì)胞中充滿了橙紅色花青素,而LPM細(xì)胞則表現(xiàn)為極淺的粉色。2、分子標(biāo)記檢測(cè)出RWT和LPM基因組多態(tài)性條帶66條,其中有18條是LPM中獨(dú)有的條帶,另外48條是LPM中缺失的,RWT和LPM間相似性系數(shù)為0.868,遺傳多態(tài)性13.2%。3、利用高效液相法測(cè)定了RWT和LPM中類黃酮成分及含量,結(jié)果表明RWT花瓣中含有的天竺葵素、飛燕草素及矢車菊素是其主要的成色物質(zhì),LPM中僅檢測(cè)到矢車菊素的積累,天竺葵素和飛燕草素的缺失是其花色變淺的根本原因。Q-PCR結(jié)果顯示LPM中花青素合成早期基因CHS、CHI表達(dá)明顯下調(diào),ANS在LPM整個(gè)花色發(fā)育過程中都保持著極低的轉(zhuǎn)錄水平,這些基因,尤其是ANS的低表達(dá)可能抑制或阻斷了花青素的生物合成,最終導(dǎo)致花瓣顏色的變化。4、通過RNA-Seq高通量測(cè)序技術(shù)建立了天竺葵花瓣轉(zhuǎn)錄組數(shù)據(jù)庫,測(cè)序共組裝獲得89817個(gè)unigene,在LPM和RWT文庫中檢測(cè)到差異表達(dá)基因1503個(gè),其中在LPM中顯著上調(diào)表達(dá)基因700個(gè),顯著下調(diào)表達(dá)基因803個(gè)。Unigene在多個(gè)公共數(shù)據(jù)庫中檢測(cè)到同源蛋白,其中在SignalP庫中比對(duì)最多。KEGG通路富集分析顯示差異基因極顯著性富集在類黃酮生物合成通路中。5、根據(jù)轉(zhuǎn)錄組測(cè)序數(shù)據(jù)預(yù)測(cè)到177個(gè)unigene可能涉及18種天竺葵花色形成基因,包括花青素合成基因,花青素修飾基因,黃酮和黃酮醇合成基因,花青素運(yùn)輸有關(guān)的基因;另外,預(yù)測(cè)到多個(gè)花色調(diào)節(jié)轉(zhuǎn)錄因子。Q-PCR驗(yàn)證14個(gè)結(jié)構(gòu)基因和3類轉(zhuǎn)錄因子的表達(dá)量,結(jié)果與轉(zhuǎn)錄組數(shù)據(jù)基本一致,基因表達(dá)以下調(diào)為主。
[Abstract]:Flower color, as an important economic index of ornamental flowers, has become the primary cultivation goal in the improvement of plant traits. In this study, a stable flower color mutant LPM. was screened out by carbon ion beam irradiation on young branches of geranium. In order to reveal the mechanism of flower color variation induced by ion beam, the causes of flower color formation were analyzed by observing the wild type and mutant phenotype of geranium. The composition and content of flavonoids and the differential expression analysis of key structural genes of flower color formation were studied systematically. The mechanism of flower color lightening after ion beam irradiation was studied systematically. The petal transcription group was sequenced deeply by RNA-Seq high throughput sequencing technique, and the differentially expressed unigene, genes between wild type and mutant were screened to predict the flower color formation of gerbils. Finally, the molecular mechanism of flower color change induced by ion beam irradiation was clarified, which provided a theoretical basis for subsequent flower color improvement and new variety selection. The main contents and results of this study are as follows: 1. A flower color mutant LPM, was screened by ion beam 30Gy irradiation and its color and physiological indexes were significantly changed compared with the control. Anatomical observation showed that RWT petal cells were filled with orange anthocyanins, while LPM cells showed very light pink. 2. 66 bands of RWT and LPM genomic polymorphism were detected by molecular markers, of which 18 were unique to LPM. The other 48 were missing from LPM. The similarity coefficient between RWT and LPM was 0.868, and the genetic polymorphism was 13.2%. The components and contents of flavonoids in RWT and LPM were determined by high performance liquid chromatography (HPLC). The results showed that germanium was contained in RWT petals. Elaeagnus angustifolia and Cypermethrin are the main color-forming substances. Only the accumulation of procyanidin was detected in LPM, and the deletion of geranium and permethrin was the root cause of the lighter flower color. Q-PCR results showed that the anthocyanin synthesis early gene CHS, in LPM was found to be the root cause of lighter flower color. Q-PCR results showed that anthocyanin synthesis in LPM was the early gene of anthocyanin synthesis. The expression of CHI was significantly down-regulated, and ANS maintained a very low transcriptional level during the whole floral development of LPM. The low expression of these genes, especially ANS, may inhibit or block anthocyanin biosynthesis and eventually lead to the change of petal color. The database of gerbils petal transcription group was established by RNA-Seq high throughput sequencing. 89817 unigene, differentially expressed genes were detected in LPM and RWT libraries, of which 1503 genes were upregulated in LPM. 803 genes were significantly down-regulated. Unigene was detected in several public databases, among which the most homologous proteins were compared in SignalP library. Kegg pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in the flavonoids biosynthesis pathway. 5, the Kegg pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in the flavonoids biosynthesis pathway. According to the sequencing data of transcriptional group, it was predicted that 17 unigene might be involved in 18 kinds of geranium flower color forming genes, including anthocyanin synthesis gene, anthocyanin modification gene, flavonoids and flavonoids alcohol synthesis gene, anthocyanin transport related genes. In addition, a number of color-regulated transcription factors were predicted. Q-PCR verified the expression of 14 structural genes and 3 kinds of transcription factors, the results were basically consistent with the data of the transcriptional group, and the gene expression was mainly down-regulated.
【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院(近代物理研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S682.19
本文編號(hào)：2483347
[Abstract]:Flower color, as an important economic index of ornamental flowers, has become the primary cultivation goal in the improvement of plant traits. In this study, a stable flower color mutant LPM. was screened out by carbon ion beam irradiation on young branches of geranium. In order to reveal the mechanism of flower color variation induced by ion beam, the causes of flower color formation were analyzed by observing the wild type and mutant phenotype of geranium. The composition and content of flavonoids and the differential expression analysis of key structural genes of flower color formation were studied systematically. The mechanism of flower color lightening after ion beam irradiation was studied systematically. The petal transcription group was sequenced deeply by RNA-Seq high throughput sequencing technique, and the differentially expressed unigene, genes between wild type and mutant were screened to predict the flower color formation of gerbils. Finally, the molecular mechanism of flower color change induced by ion beam irradiation was clarified, which provided a theoretical basis for subsequent flower color improvement and new variety selection. The main contents and results of this study are as follows: 1. A flower color mutant LPM, was screened by ion beam 30Gy irradiation and its color and physiological indexes were significantly changed compared with the control. Anatomical observation showed that RWT petal cells were filled with orange anthocyanins, while LPM cells showed very light pink. 2. 66 bands of RWT and LPM genomic polymorphism were detected by molecular markers, of which 18 were unique to LPM. The other 48 were missing from LPM. The similarity coefficient between RWT and LPM was 0.868, and the genetic polymorphism was 13.2%. The components and contents of flavonoids in RWT and LPM were determined by high performance liquid chromatography (HPLC). The results showed that germanium was contained in RWT petals. Elaeagnus angustifolia and Cypermethrin are the main color-forming substances. Only the accumulation of procyanidin was detected in LPM, and the deletion of geranium and permethrin was the root cause of the lighter flower color. Q-PCR results showed that the anthocyanin synthesis early gene CHS, in LPM was found to be the root cause of lighter flower color. Q-PCR results showed that anthocyanin synthesis in LPM was the early gene of anthocyanin synthesis. The expression of CHI was significantly down-regulated, and ANS maintained a very low transcriptional level during the whole floral development of LPM. The low expression of these genes, especially ANS, may inhibit or block anthocyanin biosynthesis and eventually lead to the change of petal color. The database of gerbils petal transcription group was established by RNA-Seq high throughput sequencing. 89817 unigene, differentially expressed genes were detected in LPM and RWT libraries, of which 1503 genes were upregulated in LPM. 803 genes were significantly down-regulated. Unigene was detected in several public databases, among which the most homologous proteins were compared in SignalP library. Kegg pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in the flavonoids biosynthesis pathway. 5, the Kegg pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in the flavonoids biosynthesis pathway. According to the sequencing data of transcriptional group, it was predicted that 17 unigene might be involved in 18 kinds of geranium flower color forming genes, including anthocyanin synthesis gene, anthocyanin modification gene, flavonoids and flavonoids alcohol synthesis gene, anthocyanin transport related genes. In addition, a number of color-regulated transcription factors were predicted. Q-PCR verified the expression of 14 structural genes and 3 kinds of transcription factors, the results were basically consistent with the data of the transcriptional group, and the gene expression was mainly down-regulated.
【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院(近代物理研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S682.19
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